The present invention relates generally to the field of medicine. More particularly, the invention relates to the field of oncology, especially the treatment of cancers of an epithelial origin.
Purine nucleosides (e.g., adenosine, deoxyadenosine, guanosine, deoxyguanosine), which comprise bi-cyclic nitrogenous purine bases (adenine, guanine) linked to a pentose sugar (ribose, deoxyribose), are found in all cell types, e.g., serving as constituent nucleosides of both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). At high concentrations, purines and their derivatives have been shown to arrest normal cell growth and cause apoptosis in certain cell types, such as endothelial cells, macrophages, and lymphocytes. It has been hypothesized that cell death usually occurs through conversion of adenosine into nucleotides (via phosphorylation) and/or into S-adenosylhomocysteine, which in turn induce pyrimidine starvation or inhibit cellular methylation. The high concentrations of adenosine necessary to cause cell death are difficult to maintain, however, due to the many cellular processes by which adenosine can be converted to other products. See Bynum, Cancer Res. 40: 2147-2152 (1980); Archer et al., J. Cell. Phys. 124:226-232 (1985); Henderson et al., Pharmac.and Ther. 8: 539-571 and 573-604 (1980).
Adenosine (Ado) is reportedly released from cells in response to alterations in oxygen supply or demand, and has been reported to be a potent vasodilator involved in the metabolic regulation of blood flow. At less than toxic concentrations, adenosine has been reported to have both cardio-protective and neuro-protective properties [Olafsson et al., Circulation, 76: 1135-1145 (1987); Dragunow and Faull, Trends in Pharmacol Sci., 9: 193 (1988).]
Adenosine deaminase (ADA) is the hydrolytic enzyme that catalyzes the deamination of adenosine and deoxyadenosine to inosine and deoxyinosine, and thus is one of the enzymes involved in controlling adenosine/deoxyadenosine levels. ADA is found at especially high levels in the spleen, thymus, and B and T lymphocytes. Adenosine monophosphate deaminase (AMP deaminase, AMPDA) is functionally related to adenosine deaminase, converting adenosine monophosphate to inosine monophosphate. ADA plays an essential role in leukocytes and its absence is associated with a severe, inherited combined immunodeficiency disease.
Interestingly, although ADA is capable of deaminating both adenosine and 2-deoxyodenosine (dAdo), it is principally dAdo that accumulates in plasma following dosing with an ADA inhibitor, such as deoxycoformycin (dCF). Apparently, deamination of adenosine occurs principally at the monophosphate level by the enzyme AMP deaminase. The dAMP moiety is a poor substrate for AMP deaminase, so deamination of dAdo is largely dependent on ADA, and ADA-inhibition results in dAdo accumulation. [Plunkett and Gandhi, Hematol. Cell Ther. 38: S67-S74 (1996).]
Inhibitors of ADA have been recognized as potential immunosuppressive agents, and many early studies of the cytotoxicity of adenosine deaminase inhibitors have involved human lymphocytes. [See, e.g., O""Dwyer et al., Annals Int. Med. 108: 733-743 (1988).] For example, dCF, a powerful ADA inhibitor (Ki for erythrocyte ADA of 2xc3x9710xe2x88x9212), has been used to treat lymphatic leukemias and is FDA-approved (Pentostatin) to treat hairy cell leukemia. Coformycin, the ribosyl analog of dCF, also acts as an ADA inhibitor. The pharmacology and efficacy of dCF and two other prominent nucleoside analogs 2-chlorodeoxyadenosine, (CDA, cladribine) and arabinosyl-2-fluoroadenine monophosphate (Fxe2x80x94araxe2x80x94AMP, fludarabine) for treating lymphoid malignancies are reviewed in Plunkett and Gandhi, Hematol. Cell Ther. 38: S67-S74 (1996), and Diliman, R., Seminars in Hematoloogy, 31: 16-27 (1994), incorporated herein by reference. The toxicity of ADA inhibitor compounds appears to relate to their causing an accumulation of toxic intracellular levels of dAdo, which (through conversion to dATP via successive phosphorylations) inhibits ribonucleotide reductase. The lethal effects of dAdo on blood cells has been extensively studied and reported in the literature.
Not all of the studies involving ADA inhibitors have focused on lymphoid malignancies. Camici et al., Int. J. Cancer, 62: 176-183 (1995) reported an assessment of the effect of deoxycoformycin (.001 to 1 xcexcM) and 2xe2x80x2-deoxyadenosine (0 to 500 xcexcM) on the growth of two cultured human colon carcinoma cell lines and on Chinese hamster ovary (CHO K-I) cells. Neither compound was reported to be toxic when used alone, whereas their combination was reported to cause cell growth inhibition, with the CHO cells more sensitive than the colon carcinoma cells. At the concentrations tested, 50-150 xcexcM deoxyadenosine was required to approach full cell growth inhibition. The authors suggest that phosphorylation of deoxyadenosine by adenosine kinase plays a central role in the toxicity of the combination therapy, and observed that the cytotoxic effect was almost completely reversed in the three cell lines when inhibitors of adenosine kinase were added to the cell culture medium. Introduction of dipyridamole to inhibit deoxyadenosine uptake also was reported to reverse the toxicity.
Svendsen et al., Cancer Chemotherapy and Pharmacology 21(1): 35-39 (1988) reported that simultaneous administration of 3xe2x80x2deoxadenosine N1-oxide and either ERNA or dCF to mice bearing Ehrlich ascites tumor cells resistant to 3xe2x80x2-deoxyadenosine N1-oxide resulted in 80-90% inhibition of tumor growth in vivo.
Rowland et al., Arch. Biochem. Biophys., 239(2): 396-403 (1985) developed a rat hepatoma cell line that was ADA-dependent and dCF sensitive. The authors observed that dCF-resistant variants developed and determined that such cells have progressively increasing concentrations of ADA activity, apparently resulting from ADA gene amplification. A dCF-resistant CHO cell line developed by the authors also demonstrated extreme increases in ADA activity, but this change was not attributable to gene amplification.
Wakade et al., J. Biol. Chem., 270(30): 17986-17992 (1995) have shown that dAdo (which increases in concentration in the presence of an ADA inhibitor) causes neuronal cell toxicity in a dose-dependent manner (maximal at 300 xcexcM). Neuronal death was correlated to a dramatic increase in the dATP content of the neurons. Nanomolar concentrations of 5xe2x80x2-Iodotubercidin (ITu) were reported to completely and dose-dependently inhibit formation of dATP and protect against toxicity of sub-millimolar concentrations of dAdo. Interestingly, neither dCF nor another ADA inhibitor [erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA)] at concentrations of 3-30 xcexcM were found to modify the toxic effects of dAdo in the neuronal model. [See also Kulkarni and Wakade, J. Neurochem., 67(2): 778-786 (1996).] Wakade et al., J. Neurochemistry 67(6):2273-2281 (December, 1996) reported that 100 xcexcM dAdo (but not adenosine) in combination with 3 xcexcM dCF was toxic to chromaffin cells. The toxicity, which was associated with dATP accumulation, was eliminated by co-culture with nanomolar concentrations of ITu.
There also exists suggestions in the literature for using AMP deaminase inhibitors for therapeutic purposes. For example, Gruber and colleagues have suggested using AMP deaminase inhibitors to treat or prevent a variety of cardiovascular and other disorders. [See U.S. Pat. Nos. 5,731,432 and 4,912,092.]
The treatment of parasitic (e.g., fungal, trypanosomal) infections with ADA inhibitors in combination with 3xe2x80x2-deoxypurine nucleosides (e.g., cordycepin) has been suggested. See McCaffrey et al., U.S. Pat. Nos. 5,679,648 and 5,663,155 and International Patent Publication No. WO 96/16664.
Extracellular adenosine and adenosine triphosphate (ATP) have also been reported to cause cytotoxicity. For example, Dawicki et al., Am. J. Physiol., 273: L485-L494 (1997) reviewed literature reporting ATP-induced apoptosis in lymphocytes, and reported that extracellular ATP, ADP, AMP, adenosine, and non-metabolized adenosine analogs [3-deaxaadenosine and Z-5xe2x80x2-fluoro-4xe2x80x2,5xe2x80x2-didehydro-5-deoxyadenosine (MDL-28842)] caused apoptosis of pulmonary artery endothelial cells. The authors concluded that the ATP metabolite adenosine was responsible for the observed toxicity, since adenosine itself and nucleotides that are degraded to adenosine caused DNA damage, whereas non-metabolizable ATP analogs (e.g., ATPxcex3S) and several adenosine metabolites did not. The extracellular ATP-induced cleavage (observed at 10 xcexcM ATP, statistically significant at 100 xcexcM) was reportedly prevented by the nucleoside transport inhibitor dipyridamole.
Bynum, J. W., Cancer Res., 40: 2147-2152 (Jul., 1980) reported that mouse melanoma cells were xe2x80x9cmoderately sensitivexe2x80x9d to adenosine, with 80% growth inhibition being observed at 50 xcexcM, compared to 5 xcexcM or 400 xcexcM reportedly required to achieve similar effects in lymphoid cells and fibroblasts, respectively. The different sensitivities were not attributed to ADA, because the lymphoid cells had two to four times the level of ADA activity than the melanoma cells or fibroblasts. The authors suggested that adenosine""s toxicity may be caused by interruption of pyrimidine biosynthesis and resultant depletion of pyrimidines. Guanosine also reportedly possessed growth-inhibitory properties. The author reported that homocysteine thiolactone (HCT) enhanced the cytotoxicity of adenosine, but not of guanosine. See also Archer et al., J. Cell. Physiol., 124: 226-232 (1985).
Homocysteine is a compound which, in high concentrations, has been identified as a prevalent risk factor for myocardial infarction and stroke. Wang et al., J. Biol. Chem., 272(40): 25380-85 (Oct., 1997) reported that homocysteine (10-50 xcexcM, a range that overlaps levels observed clinically) caused inhibition of DNA synthesis in vascular endothelial cells and arrested their growth at the G1 phase of the cell cycle, which may play an important role in the arteriosclerotic disease process. Kredich et al., Proc. Natl. Acad. Sci. USA, 76(5): 2450-2454 (1979) reported that addition of 100 xcexcM L-homocysteine thiolactone to cells treated with the ADA inhibitor EHNA and adenosine had the effect of enhancing adenosine toxicity towards a human lymphoblast cell line.
Yet another body of research has focused on use of purine nucleotides or hydrolysis-resistant purine nucleotide analogs as anti-neoplastic agents. For example, Trepel et al., U.S. Pat. Nos. 5,415,873 and 5,641,500, report that certain hormone-independent prostatic tumor cell lines express a P2 subtype purinergic receptor which interact with ATP, and that such growth of such cell lines is inhibited by ATP. Adenosine, which lacks phosphate moieties, is reported to be from about 40-fold to about 500-fold less potent an agonist than ATP of these cell types.
In U.S. Pat. No. 5,227,371, Rapaport reports that adenine nucleotide or adenosine plus inorganic phosphate, but not adenosine alone, yields a sustained xe2x80x9csecondary wavexe2x80x9d of extracellular blood plasma ATP levels. Rapaport suggests that extracellular ATP increases may have several beneficial effects, including tumor growth inhibition. In U.S. Pat. Nos. 5,049,372 and 4,880,918, Rapaport suggests that ATP or ADP can be used as selective tumor growth inhibition agents, whereas purines will inhibit the growth not only of tumor cells but also of normal cells. Rapaport""s explanation is that ADP or ATP penetrate the plasma membrane of tumor cells, but not normal cells, without degradation to AMP or adenosine. Rapaport explicitly states that the observed effects on cellular growth is unique to ADP and ATP and cannot be duplicated by adenosine.
Thus, purine nucleosides, purine nucleotides, derivatives thereof, and ADA inhibitors have been investigated by several research groups and, in some instances such as the treatment of hairy cell leukemia with dCF, have shown limited success. However, neoplastic diseases (e.g., cancer) remain one of the leading killers in modern societies, and a long felt need exists for new therapeutic regimens to treat neoplastic diseases, especially non-lymphoid related neoplastic diseases. A long felt need also exists for effective treatments which can be carefully controlled and modulated to maintain efficacy and minimize toxicity to the patient""s non-cancerous tissues and cells.
The present invention provides novel chemotherapeutic materials and methods that address one or more of the foregoing long felt needs.
In particular, the present invention provides materials and methods for the treatment of neoplastic disease states, especially cancers of cells/organs of epithelial origin. For example, the invention provides combination chemotherapy materials and methods for treatment comprising a first agent comprising adenosine or an adenosine derivative and a second agent comprising an inhibitor of at least one of the enzymes adenosine deaminase and AMP deaminase.
In one embodiment, the invention provides a composition comprising: a first compound selected from the group consisting of adenosine, adenosine derivatives, and pharmaceutically acceptable salts thereof, in admixture with a second compound selected from the group consisting of adenosine dearninase inhibitors (ADAI), adenosine monophosphate deaminase inhibitors (AMPDAI), and pharmaceutically acceptable salts thereof. Since the composition is useful for medical treatment, in a preferred embodiment it also comprises a pharmaceutically acceptable carrier. The composition may further include additional therapeutic agents, such as homocysteine compounds that potentiate the effects of the first two compounds, and/or additional agents such as preservatives and the like. ADAI and AMPDAI compounds whose inhibitory activities are characterized by sub-nanomolar inhibition constants (Kixe2x89xa61 nM) are preferred. In one preferred variation, the second compound of the composition has dual activities as both an ADAI and an AMPDAI. In another variation, the second compound is an ADAI, and the composition further includes a third compound that is an AMPDAI.
In a related embodiment, the invention provides a unit dose comprising: a first composition comprising a member selected from the group consisting of adenosine, adenosine derivatives, and pharmaceutically acceptable salts thereof, and a second composition comprising a member selected from the group consisting of adenosine deaminase inhibitors, adenosine monophosphate deaminase inhibitors, and pharmaceutically acceptable salts thereof. The compositions are preferably included in the unit dose at concentrations effective to inhibit the growth of neoplastic cells in a cancer patient.
In one variation, the unit dose is formulated wherein the first and second compositions are in admixture with each other. Preferably this mixture further includes a pharmaceutically acceptable carrier. In yet another variation, the unit dose is formulated such that the first and second compositions are packaged together as a kit, but are not in admixture. Separate packaging of the two compositions permits administration by separate routes, at separate times, and/or at separate rates, and permits formulating each composition uniquely.
In a preferred embodiment, the unit dose is packaged as kit with one or more additional compositions that are useful for enhancing the treatment regimen of a cancer patient. For example, the unit dose further includes a composition that comprises homocysteine in an amount effective to potentiate the anti-neoplastic activity of the first and second compositions.
In a highly preferred embodiment, the unit dose further includes a composition that comprises a protective agent such as a nucleoside transport inhibitors or adenosine kinase inhibitor. It is contemplated that the two or more anti-neoplastic therapeutic agents are administered via a route designed to achieve high concentrations at the site of a tumor, whereas the protective agent(s) is administered systemically to protect healthy (non-cancerous) tissues from the anti-neoplastic agents.
The invention also provides for methods of treatment that involve administration of compounds, compositions, and unit doses of the invention for the treatment of disease states, particularly neoplastic disease states. For example, the invention provides a method of treating a neoplastic disease state in a patient in need of such treatment, comprising the steps of: administering to a patient suffering from a neoplastic disease state a first compound selected from the group consisting of adenosine, adenosine derivatives, and pharmaceutically acceptable salts thereof, and a second compound selected from the group consisting of adenosine deaminase inhibitors (ADAI), adenosine monophosphate deaminase inhibitors (AMPDAI), and pharmaceutically acceptable salts thereof As explained below, treatment of malignancies of epithelial cell origin, such as those of the lungs, breasts, gastrointestinal system, genitorurinary tract, or reproductive organs is specifically contemplated. A highly preferred embodiment involves treatment of ovarian cancers.
In one variation, the method of treatment involves administering one or more of the anti-neoplastic compounds in a manner that generates high concentrations in or around the tumor area, and administering protective agents systemically to protect the patient""s healthy tissues and cells, including the heart, against toxic side-effects of the anti-neoplastic compounds.
Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, including the drawing and detailed description, and all such features are intended as aspects of the invention. Likewise, features of the invention described herein can be re-combined into additional embodiments that also are intended as aspects of the invention, irrespective of whether the combination of features is specifically mentioned above as an aspect or embodiment of the invention. Also, only such limitations which are described herein as critical to the invention should be viewed as such; variations of the invention lacking limitations which have not been described herein as critical are intended as aspects of the invention.
In addition to the foregoing, the invention includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations specifically mentioned herein. Although the applicant(s) invented the full scope of the claims appended hereto, the claims appended hereto are not intended to encompass within their scope the prior art work of others. Therefore, in the event that statutory prior art within the scope of a claim is brought to the attention of the applicants by a Patent Office or other entity or individual, the applicant(s) reserve the right to exercise amendment rights under applicable patent laws to redefine the subject matter of such a claim to specifically exclude such statutory prior art or obvious variations of statutory prior art from the scope of such a claim. Variations of the invention defined by such amended claims also are intended as aspects of the invention.